SUMMARY OF WORK: Project 1: ?-Arrestin Biased Mechanosensitive Angiotensin Receptor Signaling Mechanotransduction plays an important role in pathophysiological processes such as vascular constriction, cardiac hypertrophy, and fluid balance through osmoregulation of thirst and salt appetite. It is now appreciated that the angiotensin-II type-1 receptor (AT1R) can function as a mechanosensor in a number of cell types and does not require the ligand angiotensin II (AngII). We have recently discovered that AT1Rs when acting as mechanosensors selectively engage ?-arrestin to induce cellular signaling. During the past PPG funding period, together with Drs. Lefkowitz, Stamler and Koch, we have studied ?-arrestin-mediated signaling in the absence of G protein activation and have elucidated the scientific basis for the concept that we now term ?biased GPCR signaling. The overall objective of this project is to determine the molecular and structural basis for the transduction of AT1R signals in response to changes in membrane stretch and biased ligands, and to understand the physiological consequence of AT1Rs as mechanosensors as it relates to the regulation of thirst and salt appetite, critically important in the treatment of patients with heart failure. Our central hypothesis, is that mechanical stretch allosterically modulates the AT1R to induce a unique receptor conformation that allows for the phosphorylation of specific GRK sites on the C-terminal tail of the receptor. Phosphorylation of select amino acid residues on the c-tail of the AT1R defines a ?bar-code? that promotes the recruitment of ?-arrestin to form an AT1R-?-arrestin complex that results in a unique pattern of cellular signaling known as biased signaling. We further hypothesize that AT1Rs in cells in the brain that are involved in osmoregulation respond to osmotic stretch by activating ?-arrestin-biased AT1R signaling as a homeostatic mechanism to maintain salt and water balance in the body.